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1、芽孢杆菌筛选分离（）Experimentalmethod:Samples:purchasedbacteria,commercialmicrobialpreparations(solidorliquid),soil,waterordeadwood,rottenleaves(record,samplinglocation,PHvegetation,etc.).Medium:peptone,10g/Lbeefextract,3-5g/L,NaCl5g/L,PH7.2Enrichmentculture:Takethe5gsedimentorgutsoluble0.5
2、g,with45(50)mL/250mL(sterilewaterflaskwithaglassbead),oscillator15-20min,5mLbacterialsuspensionor5mLsamplesadding45mLbudmedium/250mLErlenmeyerflask,75DEG-80DEG15-20minwaterbathto35DEG-37DEG150-200r/minoscillation24h.Stainingfortwotothreetimes,trainingreserve.Ortake1gsediment(soil)i
3、n20mLbrothmedium,/250mLtrianglebottle,eightlayersofgauzesealing,75-8024hwaterbath,15-20min,andthen150-200r/minoscillation.Microscopicexaminationofsporulation,sporecolonieswereselectedwithcrossedculturedtoobtainsinglecolonies.Isolationandpurification:Theenrichmentofbacteriain75-80OC
4、waterbath15-20min,10-3,10-4,gradientdilutionof10-5,10-6,10-7,selectthreesuitablegradientmixedbacteriaorcoatedonnutrientagarmaltjuicemediumordirectlycoatedonthecaseinmediumscreening,canalsobecrossedseparation(crossedthefirstflatplateisarrangedonthe30C(24-48h)or37C(18-24h),sterilityt
5、est,surfacewatercondensationdrying).Eachgradientistwoplates,andtheplateisinvertedat35-3724-48h.Thesinglecolonieswerenumberedandthecolonieswerefastgrowing,thesurfacewasdryandrough,andtheopaquecoloniesweretransferredtotheslope.Takealittlesandsmear,dosporestaintodeterminewhetherbacill
6、us.Screeningofproteaseproducingstrains:Operatingprocedures:sampledilution-heattreatment(75-80C)--gradientdilutionspreadplatesinglecolonynumber(transferinclined)-microscopy(sporestainingsmear)-screening(pick-caseinflatsieve(pick)enzymeproductionmedium,centrifugedsupernatantproteinen
7、zymeactivityassay)transfer-slantpreservation.Generally,theselectedstrainsaredry,roughandopaque,astargetbacteria.Todeterminethecolonyatbacillus,respectively,werealittlelawnmadeofbacterialsuspension,orcrossedproperlydilutedcoatingonthecaseinplate,35DEG-37DEG24-48h,transparentcirclear
8、oundthecolony,wereC/Hbacte
